EVALUATION OF A KETOGENIC FORMULATION FOR TREATING PATIENTS WITH ACUTE BRAIN INJURY Hayden Thomas Wesley White MBBCH MMED (Wits) FCICM FRACP A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Faculty of Medicine Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury Abstract Acute brain injury (ABI), including cerebral vascular accidents (CVA) and traumatic brain injury (TBI), are relatively common diseases which may lead to death, significant morbidity and place a heaving burden on health care systems globally, both in suffering and cost. Evidence has emerged from basic science research that ketones (beta-hydroxybutyrate and acetoacetate) which are water-soluble molecules produced by the liver during times of starvation, may have neuroprotective effects. It is therefore theoretically possible that increasing plasma ketone concentrations via intravenous or oral supplementation, may lead to improved outcomes following ABI. This thesis documents a systematic investigation into the possibility that ketone supplementation may lead to benefits in patients with a variety of chronic and acute neurological conditions. While the main emphasis is on ABI, other conditions where ketones may be useful are also reviewed. Beginning with a review of ketones, their potential mechanisms of action and likely neuroprotective roles in disease, this thesis examines the effects of a novel hypertonic intravenous ketone solution on plasma, cerebrospinal fluid (CSF) and brain ketone concentrations in animals, then looks at the hurdles in producing such an intravenous solution as opposed to enteral formulations for humans, before investigating baseline ketone concentrations in patients with ABI and finally culminating in an interventional study where a modified enteral ketone supplement is administered to patients with ABI to determine whether adequate plasma ketone concentrations are attainable. The animal study performed in rats involved the administration of hypertonic saline/ketone solutions of varying concentrations to anaesthetised rats. The results demonstrated that both CSF and beta-hydroxybutyrate (BHB) concentrations increased over time (p<0.0001) and that this was dependent on the BHB concentration in the solution (p<0.0001). Measurement of BHB concentrations in brain tissue noted a dose dependent increase compared to control with the highest concentration almost double 0.15 mmol/l versus 0.28 mmol/l. Significantly, the infusions did not lead to a metabolic acidosis which has been a concern amongst researchers. These findings led to an attempt to produce an intravenous solution for administration to humans. However, the cost of producing this formulation proved prohibitive. Another source i Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury of ketones was therefore sort. While this was underway, as baseline data was lacking, we undertook a study to measure ketone concentrations in patients with ABI. The subsequent study recruited 38 patients with ABI and followed them for up to 7 days. Both plasma and CSF (where possible) concentrations of BHB and Acetoacetate were measured on a daily basis. 22 patients completed the full 7 days of observations, 7 CVA, 7 subarachnoid haemorrhage (SAH) and 8 TBI. During the study period, BHB concentrations increased initially but normalized by day 3 while acetoacetate (AcAc) concentrations remained within the normal range. There was a weak correlation between blood and CSF BHB (Spearman’s rho = 0.62, P=0.054). It was therefore clear that as blood ketone concentrations remained low, an external source would be required to increase blood ketone concentrations to clinically relevant concentrations. Therefore, a study investigating enteral based supplementation with the view to increasing ketone concentrations in ABI patients was designed. The study was to look at the feasibility of administering a ketogenic enteral formulation to patients with ABI over a 6 day period. Outcomes included ketone concentrations in blood and CSF (where possible) and monitoring for adverse events resulting from the ketone administration. It was determined that the commercially available product Ketocal (Nutricia) would be the most suitable enteral supplement. However, as this product was lacking in sufficient protein, a protein supplement was added. The study was entitled “Induction of ketogenesis in patients with acute brain injury via oral administration of a ketogenic feed” or KABI and intended to recruit 20 patients. While the KABI study was under development, 2 further reviews were undertaken in order to further examine the current literature on cerebral nutrition and the place of ketogenesis in this process. The first reviewed the place of metabolic substrates other than glucose on cerebral energetics. These included ketones, lactate, non-esterified fatty acids, branch- chain amino acids, and TCA cycle intermediates including triheptanoin. It was noted that while glucose is the main substrate for the brain in health, other metabolic substrates may prove beneficial in the injured brain. While ketones have received the most in-depth study and appear to be an excellent substitute for glucose, exploration into the field of cerebral energetics is in its infancy and further research is necessary. The second was a systematic review of publications focusing on the use of ketones in both acute and chronic neurological disorders in adults (excluding chronic poorly controlled epilepsy). As expected, the data is very limited, consisting of mainly case series and small ii Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury randomised controlled trials. Study subjects included Alzheimer’s disease, severe refractory status epilepticus, intracranial neoplasms, TBI and Parkinson’s disease. Although outcomes were largely positive, the poor quality of the evidence prohibits firm recommendations other than that further research is necessary to explore the potential benefits of ketones in a number of neurological conditions. The KABI study was subsequently undertaken and completed. We recruited 20 patients, 5 females and 15 males, 3 with stroke, 2 with subarachnoid haemorrhage and 15 with traumatic brain injury. We were able to demonstrate a significant increase in both plasma beta-hydroxybutyrate and acetoacetate to 0.61 ± 0.53 mmol/l (p =0.0005) and 0.52 ± 0.40 mmol/l (p<0.0001) over the 6 day period. The total daily Ketocal® caloric intake was positively correlated with plasma beta-hydroxybutyrate concentrations (p=0.0011). The feeds were well tolerated in 19 out of the 20 patients with no clinically significant changes in acid/base status over the 6 days with pH remaining within normal range. One patient developed a metabolic acidosis and feeds were ceased. We concluded that in patients with acute brain injury, although an enterally administered ketogenic formulation was well tolerated more work was required to determine the optimal concentration of ketones necessary to impact on cellular energetics and the best means of inducing ketosis. One of the main issues limiting progress in investigating ketones as a therapeutic tool is the difficulty in attaining high plasma ketone concentrations rapidly and consistently using the current enteral approach. The ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate, may provide a solution to this problem. The thesis concludes by highlighting gaps in knowledge, challenges and avenues for future research. iii Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co- authors for any jointly authored works included in the thesis. iv Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury Publication during candidature Peer Reviewed Manuscripts: 1. White H, Venkatesh B. Clinical review: ketones and brain injury. Crit Care. 2011;